PROJECT SUMMARY/ABSTRACT Understanding responsive mechanisms of metalloproteins is key to elucidate biological functions of copper (Cu) and to identify the causes of diseases resulting from abnormal metal homeostasis. The cellular Cu uptake and secretion require relevant metalloproteins to adjust in a spatiotemporally coordinated manner to assure proper cellular Cu level. However, in the Cu field, little is known about how metalloproteins are individually regulated nor systematically cooperate with each other in their native environment, i.e., in cells. Our research goal is to understand the responsive mechanisms of Cu-uptake and secretory metalloproteins in live mammalian cells, with specific focuses on how metalloproteins adjust their behaviors such as spatial distributions, oligomeric states, inter-protein and inter-domain interactions for proper Cu balance in a spatiotemporally defined manner. Previous achievements of the PI include discoveries of novel mechanisms of MerR-family metalloregulators in regulating transcription and Cu-responsive dynamic assembly of efflux pumps by examining the protein-DNA interaction and protein diffusive behaviors in live bacteria using single-molecule super-resolution microscopy. Leveraging the power of these technologies, in combined with the recently developed live-cell single-molecule fluorescence- resonance-energy-transfer assay, we will elucidate the responsive mechanisms of metalloproteins in the uptake and secretory pathways in live mammalian cells. Using CTR1 and ATOX1-ATP7A/B as the initial examples of uptake and secretory metalloproteins, the proposed experiments will (1) quantify Cu-dependent oligomeric state distribution and identify the Cu-responsive moiety of CTR1; (2) define the preferential interaction of ATOX1 to ATP7A and ATP7B and how mutations in ATP7B affect Cu homeostasis in cellular Cu defending using induced pluripotent stem cells derived hepatocytes. In addition to primary approaches of single-molecule super-resolution fluorescence imaging techniques, complementary bulk spectroscopic and biochemical measurements will be compared. The research program is further enhanced by collaborations with the experts in Cu homeostasis and stem cell fields. The research is significant because it will provide mechanistic insights into metalloprotein- mediated Cu-uptake and secretion processes as well as complementary information for synchrotron X-ray fluorescence studies on intracellular Cu-redistribution. The comparison between human induced pluripotent stem cell (hiPSC)-derived healthy and diseased hepatocytes will inform how disease mutations disrupt cellular Cu balance, providing the knowledge base to devise therapeutic strategies for Wilson's diseases. The research is innovative because it represents a substantive departure from the status quo by shifting focus to define response mechanisms of metalloproteins using advanced approaches including single-molecule super-resolution microscopy and hiPSC-derived hepatocytes. The live-cell imaging approach also circumvents the general challenge in studying membrane complexes, whose in vitro reconstitution is technically demanding. The hiPSC- derived diseased hepatocytes provide an ideal platform to study the pathogenesis of Wilson's disease.